Target-dependent transcription using deletion mutants of N4 RNA polymerase

• US 7,838,270 B2
• Filed: 12/23/2003
• Issued: 11/23/2010
• Est. Priority Date: 05/22/2001
• Status: Expired due to Fees

• Current Assignee: Epicenter Technologies, University of Chicago
• Original Assignee: University of Chicago
• Inventors: Rothman-Denes, Lucia B., Davydova, Elena K., Kazmierczak, Krystyna Maria
• Primary Examiner(s): Kim; Young J
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US10/743,975
• Publication Number:
  US 20040191812A1
• Time in Patent Office: 2,527 Days
• Field of Search: None
• US Class Current: 435/91.21
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance. The method consists of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process.

Claims

1. A method of making RNA comprising:

(a) obtaining a N4 virion RNA polymerase consisting of a transcriptionally active mini-vRNAP, wherein said mini-vRNAP consists of a sequence at least 95% identical to SEQ ID NOS;

4, 6, or 8;

(b) obtaining a single-stranded DNA oligonucleotide wherein said single-stranded DNA oligonucleotide contains a N4 virion RNA polymerase promoter sequence;

(c) admixing said N4 virion RNA polymerase and said single-stranded DNA oligonucleotide; and

(d) culturing said N4 virion RNA polymerase and said single-stranded DNA oligonucleotide under conditions effective to allow RNA synthesis.